The goal of the proposed research is determine how competition at the level of host population immunity drives genetic diversification of microbial pathogens-changes that underlie shifts in disease pattern and are fundamental to the understanding of how new pathogens emerge. Specifically, we are focused on microbial pathogens that employ gene conversion as a mechanism to generate antigenic variants, evade clearance by the immune system, and persist within mammalian host reservoirs. These pathogens generate within-host antigenic variants using unidirectional recombination of variant donor alleles into expression sites-resulting in newly expressed variants with retention of the unchanged donor alleles within the genome. There is strong selective pressure for sufficient diversity among the variant alleles in the genome to evade clearance and persist, as within host persistence dramatically increases the likelihood of successful ongoing transmission. We have identified a second strong selective pressure that drives allelic diversification among strains of a pathogen. Penetration of a new strain into a region with a high prevalence of infection and consequent broad immunity against the variants encoded by an existing endemic strain requires that the new strain encode variants that will be immunologically novel and allow establishment of infection. This pressure is hypothesized to favor continual genetic probing for selective advantage and to result in the genetic change that underlies shifts in transmission and disease patterns. Understanding how this occurs in bacteria and protozoa, complex pathogens that cannot rely on mutation and the large progeny burst size as do viruses, and the consequences for transmission within endemic zones is the goal of this project. Using Anaplasma marginale as a model in which we can study pathogen infectivity and transmission in a natural mammalian host, we will determine how an emergent strain acquires competitive advantage. We propose that this occurs in a two step process: non-tandem duplication of an existing allele within the genome followed by recombination among alleles to generate diversity in the duplicated allele. In the first part of the project, we will test this model and determine the degree of change needed to encode a variant able to escape broad population immunity. In the second part, we will examine the "fitness cost" of this genomic change and determine if it underlies the ability of an emergent strain to succeed within the pathogen population. PUBLIC HEALTH RELEVANCE Significance Understanding how microbial pathogens undergo genetic change under natural selective pressures represents a major gap in knowledge in emerging infectious diseases research and is specifically relevant to public health as genetic change underlies shifts in disease phenotype, including new patterns of transmission, gain or loss of virulence, and adaptation to new host species. The proposed research specifically addresses this genetic change in a vector-borne rickettsial pathogen that is naturally transmitted among wild and domestic animals as reservoir hosts. Analysis of emerging infectious diseases reveals that >60% of emerging human diseases have their origin in an animal reservoir, >50% are caused by bacterial or rickettsial pathogens, and >25% are vector-borne pathogens (Global trends in emerging infectious diseases, Nature 451:990-995, 2008;Global Infectious Diseases Surveillance and Detection, Institute of Medicine of the National Academies, 2007). Consequently the proposed research is broadly applicable to understanding and thus better tracking and prevention of emerging disease.